This study aimed to investigate the efficiency of antimicrobial photodynamic therapy (aPDT) on Streptococcus mutans biofilm in the oral cavity using the photosensitizer chloroaluminum phthalocyanine encapsulated in chitosan nanoparticles (ClAlPc/Ch) at three preirradiation times. Biofilms of Streptococcus mutans strains (ATCC 25,175) were cultivated on bovine tooth blocks and exposed to a 10% sucrose solution three times a day for 1min over three consecutive days. The samples were randomly distributed into five treatment groups (n = 5): (I) aPDT with ClAlPc/Ch with a preirradiation time of 5min (F5), (II) aPDT with ClAlPc/Ch with a preirradiation time of 15min (F15), (III) aPDT with ClAlPc/Ch with a preirradiation time of 30min (F30), (IV) 0.12% chlorhexidine digluconate (CHX), and (V) 0.9% saline solution (NaCl). After treatment, the S. mutans biofilms formed on each specimen were collected to determine the number of viable bacteria (colony-forming units (CFU)/mL). Data were analyzed for normality using the Shapiro-Wilk test and the analysis of variance (ANOVA) and Tukey HSD tests to analyze the number of viable bacteria (α = 0.05). The one-way ANOVA showed a difference between the groups (p = 0.0003), and the Tukey HSD posttest showed that CHX had the highest microbial reduction of S. mutans, not statistically different from the F5 and F15 groups, whereas the NaCl group had the lowest microbial reduction statistically similar to the F30 group. The results demonstrate that aPDT mediated by ClAlPc/Ch when used at preirradiation times of 5-15min can be an effective approach in controlling cariogenic biofilm of S. mutans, being an alternative to 0.12% CHX.